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Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μM). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca(2+) ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca(2+) binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca(2+) citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.
Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.
BACKGROUND - Recent in vitro studies have shown that disturbed flow and oxidative conditions induce the expression of bone morphogenic proteins (BMPs 2 and 4) in cultured endothelial cells. BMPs can stimulate superoxide production and inflammatory responses in endothelial cells, raising the possibility that BMPs may play a role in vascular diseases such as hypertension and atherosclerosis. In this study, we examined the hypothesis that BMP4 would induce hypertension in intact animals by increasing superoxide production from vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and an impairment of vasodilation responses.
METHODS AND RESULTS - BMP4 infusion by osmotic pumps increased systolic blood pressure in a time- and dose-dependent manner in both C57BL/6 mice (from 101 to 125 mm Hg) and apolipoprotein E-null mice (from 107 to 146 mm Hg) after 4 weeks. Cotreatment with the BMP antagonist noggin or the NADPH oxidase inhibitor apocynin completely blocked the BMP4 effect. In addition, BMP4 infusion stimulated aortic NADPH oxidase activity and impaired vasorelaxation, both of which were prevented either by coinfusing noggin or by treating the isolated aortas with apocynin. BMP4, however, did not cause significant changes in maximum relaxation induced by the endothelium-independent vasodilator nitroglycerin. Remarkably, BMP4 infusion failed to stimulate aortic NADPH oxidases, increase blood pressure, and impair vasodilation responses in p47phox-deficient mice.
CONCLUSIONS - These results suggest that BMP4 infusion induces hypertension in mice in a vascular NADPH oxidase-dependent manner and the subsequent endothelial dysfunction. We suggest that BMP4 is a novel mediator of endothelial dysfunction and hypertension and that noggin and its analogs could be used as therapeutic agents for treating vascular diseases.
OBJECTIVE - We have previously demonstrated that endothelial xanthine oxidase (XO) levels are dependent on the NADPH oxidase. We postulated that H2O2 may modulate the irreversible conversion of xanthine dehydrogenase (XDH) to XO and sought to examine mechanisms involved.
METHODS AND RESULTS - H2O2 (100 micromol/L) decreased bovine aortic endothelial cell (BAEC) XDH protein expression, and metabolic labeling studies indicated that H2O2 stimulated conversion of XDH to XO. The decline in XDH was mimicked by the reactive oxygen species (ROS) generating compounds SIN-1 and Menadione, as well as by stimulating BAECs with angiotensin II (200 nmol/L). BAPTA-AM prevented the decline in XDH by H2O2, indicating that it was calcium-dependent. In keeping with calcium acting downstream of H2O2, the calcium ionophore A23187 (1 micromol/L) caused XDH-to-XO conversion, and this was not prevented by the antioxidants. In addition, XDH-to-XO conversion was blocked by 2-APB and NO donors and induced by thapsigargin and M-3M3FBS, implicating phospholipase C and endoplasmic reticulum calcium stores in this process.
CONCLUSIONS - Endothelial XO and XDH expression are strongly dependent on H2O2 and calcium. Stimulation of XDH conversion to XO may represent a feed-forward mechanism whereby H2O2 can stimulate further production of ROS.
Despite intracellular L-arginine concentrations that should saturate endothelial nitric oxide synthase (eNOS), nitric oxide production depends on extracellular L-arginine. We addressed this 'arginine paradox' in bovine aortic endothelial cells by simultaneously comparing the substrate dependence of L-arginine uptake and intracellular eNOS activity, the latter measured as L-[3H]arginine conversion to L-[3H]citrulline. Whereas the Km of eNOS for L-arginine was 2 microM in cell extracts, the L-arginine concentration of half-maximal eNOS stimulation was increased to 29 microM in intact cells. This increase likely reflects limitation by L-arginine uptake, which had a Km of 108 microM. The effects of inhibitors of endothelial nitric oxide synthesis also suggested that extracellular L-arginine availability limits intracellular eNOS activity. Treatment of intact cells with the calcium ionophore A23187 reduced the L-arginine concentration of half-maximal eNOS activity, which is consistent with a measured increase in L-arginine uptake. Increases in eNOS activity induced by several agents were closely correlated with enhanced L-arginine uptake into cells (r = 0.89). The 'arginine paradox' may be explained in part by regulated L-arginine uptake into a compartment, probably represented by caveolae, that contains eNOS and that is distinct from the bulk cytosolic L-arginine.
In mammalian cells, gene regulation by amino acid deprivation is poorly understood. Here, we examined the signaling pathways involved in the induction of the C/EBP homologous protein (CHOP) by amino acid starvation. CHOP is a transcription factor that heterodimerizes with other C/EBP family members and may inhibit or activate the transcription of target genes depending on their sequence-specific elements. Amino acid deficiency, when accompanied by insulin-like growth factor I signaling, results in the accumulation of CHOP messenger RNA and protein in AKR-2B and NIH-3T3 cells. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 are able to block CHOP induction in response to amino acid deprivation. Rapamycin is also able to abrogate CHOP expression, suggesting that the mammalian target of rapamycin is involved in CHOP induction by amino acid deficiency. LY294002 and rapamycin are also able to block CHOP induction by hydrogen peroxide, but do not affect expression induced by sodium arsenite or A23187. This is the first evidence that the insulin-like growth factor I/phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway is required for gene regulation by amino acid deprivation and that this pathway is involved in the induction of CHOP by both amino acid deficiency and oxidative stress by hydrogen peroxide.
Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.
Nitric oxide (NO) and superoxide are both constitutive products of the endothelium. Because NO is readily inactivated by superoxide, the bioactivity of endothelium-derived NO (EDNO) is dependent on local activity of superoxide dismutase (SOD). We examined the effects of chronic inhibition of copper-zinc SOD (CuZnSOD) using a rat model of dietary copper restriction. Male weanling Sprague-Dawley rats were fed a Cu-deficient diet and received either no Cu replacement (Cu-deficient) or Cu in the drinking water (Cu-sufficient). Compared with Cu-sufficient animals, Cu-deficiency was associated with a 68% reduction in CuZnSOD activity and a 58% increase in vascular superoxide as estimated by lucigenin chemiluminescence (both P < .05). Compared with Cu-sufficient animals, arterial relaxation in the thoracic aorta from Cu-deficient animals was 10-fold less sensitive to acetylcholine, a receptor-dependent EDNO agonist, but only 1.5-fold less sensitive to A23187, a receptor-independent EDNO agonist, and only 1.25-fold less sensitive to authentic NO (all P < .05). In contrast, acute inhibition of CuZnSOD with 10 mM diethyldithiocarbamate produced a more uniform reduction in sensitivity to acetylcholine (8-fold), A23187 (10-fold), and NO (4-fold; all P < .001). Cu-deficient animals demonstrated a 2.5-fold increase in plasma-esterified F2-isoprostanes, a stable marker of lipid peroxidation, that correlated inversely with arterial relaxation to acetylcholine (R = -.83; P < .0009) but not A23187 or authentic NO. From these findings, we conclude that chronic inhibition of CuZnSOD inhibits EDNO-mediated arterial relaxation through two mechanisms, one being direct inactivation of NO and the other being lipid peroxidation that preferentially interrupts receptor-mediated stimulation of EDNO.
Arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE) are reported to induce reinitiation of meiosis in oocytes of the surf clam Spisula sachalinensis from the Sea of Japan (Varaksin et al., Comp. Biochem. Physiol. 101C, 627-630 (1992). As the Atlantic surf clam Spisula solidissima is a commonly used model for the study of meiosis reinitiation, we examined these cells for the possible occurrence of lipoxygenases and for the bioactivity of the products. Incubation of [14C]arachidonic acid with homogenates of S. solidissima oocytes led to the formation of two major metabolites: 5R-HETE, a novel lipoxygenase product, and 8R-HETE. The products were identified by HPLC, uv spectroscopy, and GC-MS. The corresponding hydroperoxy fatty acids, the primary lipoxygenase products, were isolated from incubations of ammonium sulfate fractionated oocyte cytosol. Arachidonic and eicosapentaenoic acids were identified as constituents of S. solidissima oocyte lipids and the free acids were equally good lipoxygenase substrates. We examined the activity of C18 and C20 polyunsaturated fatty acids and their lipoxygenase products on meiosis reinitiation in Spisula solidissima oocytes, using serotonin and ionophore A23187 as positive controls. The fatty acids and their derivatives were inactive. We conclude that in the surf clam, (as in starfish), there are responding and non-responding species in regard to the maturation-inducing activity of the oocyte lipoxygenase products, and that the lipoxygenase has another, as yet uncharacterized, function in oocyte physiology.
Mannan, a ligand for the mannose/N-acetylglucosamine (GlcNAc) receptor, induces suppression of oxygen consumption and increases glucose production in the perfused rat liver, and repeated infusion of mannan causes desensitization of the responses. In this study, we examined whether activation of Kupffer cells by endotoxin and phorbol ester alters the glycogenolytic responses to mannan. Infusion of lipopolysaccharide (LPS, 10 micrograms/ ml) in the perfusate failed to inhibit the responses to mannan. Intravenous administration of LPS (1 mg/kg) 6 and 24 h before perfusion did not desensitize the responses to mannan, suggesting that the responses through mannose/GlcNAc receptors in the liver are retained even after activation of Kupffer cells by LPS. In contrast, prior infusion of phorbol 12-myristate 13-acetate (PMA, 100 nM) in vitro abolished the glycogenolytic responses to subsequently infused mannan, but not that to norepinephrine (100 nM), while prior infusions of 4-alpha-phorbol 12,13-didecanoate (100 nM), A23187 (50 nM), or forskolin (1 microM) had no effect on the mannan-induced responses. H-7, an inhibitor of protein kinase C, reduced the glycogenolytic responses to mannan, while it failed to restore the desensitization. These results suggest that protein kinase C may be involved in the process of glycogenolysis by mannan, but is unlikely to be involved in the homologous desensitization of the responses.